The present invention relates to the field of plant molecular biology. More particularly, the present invention relates to the isolation of nucleic acids encoding terpene synthases (TPSs), including a novel, multifunctional TPS identified herein as CoTPS2.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Plants emit a large group of phytogenic volatile organic compounds (VOCs) for their defense against pathogens, parasites and herbivores and for attracting pollinators (Kessler and Baldwin, 2001; Dudareva et al., 2013). VOCs are synthesized in all plant organs such as flowers, stems, leaves, roots, fruits and seeds, but the quantity and diversity of VOCs change in response to environmental stimuli (Dudareva et al., 2013). VOCs are major components of floral scent in a wide range of flowers (Knudsen et al., 1993). Over 1,700 floral VOCs have been identified from 90 different plants, and they are assumed to have functions in both attraction of pollinators and defence against pathogens (Knudsen et al., 2006; Muhlemann et al., 2014). Given the role of VOCs, the production and emission of VOCs are highly regulated spatially and developmentally. Floral VOCs are mainly composed of terpenoids, phenylpropanoids/benzenoids and volatile fatty acid derivatives which are derived from different biosynthetic routes in plants (Muhlemann et al., 2014). Terpenoids, also referred to as isoprenoids, are the largest and most diverse class of VOCs in plants (Dudareva et al., 2013). Terpenes are synthesized from two distinct and compartmentally separated pathways, the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways (McGarvey and Croteau, 1995). Phenylpropanoids and benzenoids class of metabolites are primarily derived from the carbon skeleton of phenylalanine which is produced by the shikimate pathway (Vogt, 2010; Orlova et al., 2006).
Terpene synthases (TPSs) are responsible for generating the immense diversity in terpenes produced by plants (McGarvey and Croteau, 1995). Many TPSs have the ability to synthesize multiple products from a single prenyl diphosphate substrate (Degenhardt et al., 2009). Based on the sequence relatedness and functional assessment, the TPS gene family has been divided into seven subfamilies designated TPS-a through TPS-g (Bohlmann et al., 1998; Lee and Chappell, 2008; Martin et al., 2010). TPS-a subfamily typically contains angiosperm-specific sesqui-TPSs, whereas angiosperm mono-TPSs form the TPS-b subfamily. The TPS-b subfamily contains the arginine-tryptophan motif, R(R)X8W (SEQ ID NO:1) which plays a role in the RR-dependent isomerization of GPP (Martin et al., 2010). Another angiosperm mono-TPS subfamily, the TPS-g contains members of mono-TPSs that lack the R(R)X8W (SEQ ID NO:1) motif characteristic. These TPSs produce acyclic monoterpenes that contribute to the floral VOCs (Dudareva et al., 2013). TPS-c and TPS-e subfamilies consist of angiosperm di-TPSs responsible for gibberellic acid (GA) biosynthesis namely copalyl diphosphate synthases (CPS) and kaurene synthases (KS). The different mono-, sesqui-, and di-TPS genes for synthesis of conifer-specialized terpenes belong to the gymnosperm-specific TPS-d subfamily (Martin et al., 2004). TPS-f includes the monoterpene linalool synthase of the genus Clarkia (Dudareva et al., 1996).
Cananga odorata, commonly called ylang ylang is a tropical evergreen tree of the Annonaceae family that produces fragrant flowers and is widely cultivated throughout Southeast Asia. Essential oils obtained by steam distillation from mature fresh ylang ylang flowers are used in the cosmetic industry as major components of perfumes and fragrances, in the food industry as ingredients of aromas and flavours, and in the pharmaceutical industry as active components of antibacterials and in aromatherapy (Benini et al., 2010; Burdock and Carabin, 2008; Gaydou et al., 1986). The chemical composition of floral VOCs produced by ylang ylang varieties has been previously reported (Gaydou et al., 1986; Benini et al., 2010, 2012; Brokl et al., 2013). These papers show the presence of volatile terpenes, benzenoid and phenylpropanoids in floral VOCs. Gaydou et al., described the composition of essential oils of ylang ylang flowers originating from Madagascar (Cananga odorata Hook Fil. et Thomson forma genuina). These authors found that the primary component was the monoterpene linalool (19%), and the other major compounds were two sesquiterpenes, β-caryophyllene (10.7%) and germacrene D (10.3%) (Gaydou et al., 1986). Additionally, this variety of ylang ylang from Madagascar contained more than 20% of other aromatic compounds such as p-methylanisole, benzyl benzoate, methyl benzoate and benzyl salicylate (Gaydou et al., 1986). Cananga odorata var. fruticosa or dwarf ylang ylang is another variety which is popularly grown in Southeast Asia as a small and compact shrub with highly scented flowers. Its essential oil is also used in the perfume industry. Despite the economic and social importance of this species, the biosynthetic pathways leading to the production of the floral scent of ylang ylang have not been fully understood.